Genomic characterization and evolutionary analysis of three bovine viral diarrhea virus 1b strains from Taiwan, highlighting genomic evidence of cytopathic evolution

Fig. 1. Histopathologic features of the two cattle. A dairy cow (BV236) and a beef cow (BV27) were included in this study. Gross lesions (A, C, and E) and corresponding histopathologic changes with H&E staining (B, D, and F) were recorded. Panels A and B show ileal lesions, and panels C and D show lung lesions in BV236. There were reddish plaque and rugal folds like (arrow) thinned ileal wall (A). Low-power photomicrograph of (A) illustrate lymphoid depletion, crypts ectatic and invade into the submucosa and underlying Peyer’s patches of the ileum (B). The majority of the lung parenchyma was mottled red (C). Alveolar septa and perivascular interstitium were infiltrated by small numerous of lymphocytes, plasma cells, and fewer neutrophils (D). Panels E and F illustrate lung lobes in BV27. Approximately 30–40% of the lung parenchyma in the cranioventral regions was dark red, firm and consolidation with multifocal to coalescing, well-demarcated and variably sized yellow-white nodules (E). The bronchioles and surrounding alveoli were obliterated by large numbers of neutrophils, cellular debris, fibrillary fibrin, and admixed with clusters of basophilic bacterial colonies (F). The…

Fig. 2. Whole-genome structure of two Taiwanese BVDV strains. A The BV27 strain has a genome length of 12,207 bp, comprising an 11,694 bp coding region flanked by a 371 bp 5′ untranslated region (UTR) and a 142 bp 3′ UTR. In contrast, the BV236 strain has a genome length of 15,047 bp, including a 14,493 bp coding region, a 372 bp 5′ UTR, and a 182 bp 3′ UTR (B). BV236 harbors a 2,559 bp duplication of the NS3-NS4A-partial NS4B region (highlighted in pink) and a 240 bp insertion corresponding to a partial bovine ubiquitin C gene (ubi#), highlighted in yellow

Fig. 3. Verification of host ubiquitin C sequence insertion in the BV236 genome. A Schematic representation of the genetic structure of BV236. A partial ubiquitin C coding sequence (UBC CDS) was aligned to the BV236 polyprotein using Geneious Prime. The UBC CDS is inserted within the duplicated region spanning NS3–NS4A–partial NS4B, specifically located between partial NS4B and NS3. B Validation of the insertion by PCR using primers targeting the flanking regions in NS4B and NS3. A single amplicon of the expected size (379 bp) was obtained, as indicated by the arrow. C Sanger sequencing confirmed the inserted fragment. The chromatogram shows the inserted ubiquitin C sequence, along with the flanking nucleotide regions encoding the amino acid motifs SRGG (upstream) and GPAV (downstream, within the NS3 region) in the BV236 genome

Fig. 4. Validation of BV236 genotypes by long-range PCR. A Schematic of the PCR strategy. To determine whether BV236 represented a co-infection, PCR was performed with primers targeting unique viral regions (NS2 and NS4B; blue arrows), designed to amplify the genomic region containing the viral duplication and host-derived insertion. B PCR results. Two amplicons of approximately 5.7 kb and 2.9 kb were obtained. The corresponding genomic features represented by the two products are illustrated. M: DNA marker; Mock: RNA extracted from uninfected MDBK cells

Fig. 5. Subtype identification of Taiwanese BVDV strains BV236 and BV27 through phylogenetic analysis. A Phylogenetic tree based on the nucleotide sequences of the 5′ untranslated region (UTR), and (B) Tree based on the complete polyprotein coding sequences. A total of 180 BVDV-1 strains were analyzed, including three Taiwanese isolates: BV27 and BV236 (identified in this study) and TW18H17 (previously reported). Phylogenetic trees were constructed using the maximum likelihood method implemented in IQ-TREE v1.6.12. Branch support values were assessed via 1,000 bootstrap replicates. The three Taiwanese strains are highlighted in red

Fig. 6. Pairwise comparison of polyprotein and individual viral protein sequences among Taiwanese BVDV strains. Pairwise amino acid sequence similarity and identity of the 12 individual viral proteins and the full-length polyprotein (FL) were compared between BV27 and BV236 (A), as well as between each of these and the previously reported Taiwanese strain 18H17 (B). Analyses were conducted using the BLOSUM62 substitution matrix with a similarity threshold of 1. Note: “FL-ex” refers to the polyprotein of BV236 excluding the insertion and duplicated regions

Fig. 7. Haplotype network analysis of BVDV-1b identified in Taiwan. The genetic relationships and evolutionary pathways of three Taiwanese BVDV-1b strains—18H17 (isolated in 2018) and two recent strains, BV27 and BV236 (identified in 2023)—were inferred using haplotype network analysis. Sequences of the E2 gene (A) and the NS2-3 gene (B) of 41 BVDV-1b strains originating from nine countries were included in the analysis. Haplotype networks were constructed using Minimum spanning networks implemented in PopART v1.7. Circle colors indicate the geographic origin of each haplotype, while circle size represents the number of strains within each haplotype